Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011

RECOMBINANT ANTIBODIES AND THE PHAGE DISPLAY TECHNOLOGY

Éva CsőszMolecular Therapies - Lecture 7

Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011

The aim of lecture 7 is to present the possibilities for therapeutic antibody production, to highlight the pros and cons of the different production methods. In this lecture the production of antibodies in the body and by different techniques like in hybridoma cells or the generation of high antibody diversity by phage display technology will be discussed.

Chapters in lecture 7.

7.1. IntroductionVI.I.1. The structure of antibodies and their production in the bodyVI.I.2. Antigen-antibody binding

7.2. The production of therapeutic antibodiesVI.II.1. The production of antibodies in hybridoma cells. VI.II.2. Humanized antibodiesVI.II.3. Production of human antibodies

7.3. Generation of antibodies by phage display VI.III.1. The phage display technologyVI.III.2. Generation of phage libraries

7.4. Administration of therapeutic antibodies

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Heavy chain: constant region, variable region

Light chain: constant region, variable region

Hinge region

Supervariable region

Disulfide bonds

COO-

COO-

COO-

COO-

NH3+NH3

+

NH3+

NH3+

VHVH

VLVL

CH3

CLCL

CH2

CH1

CH1

Fab region

Fc region

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The structure of antibodies

VH1 VH2 DHnDH1VHnVH4VH3 JH1 CCµJHnJH3JH2 C C C α

JH2DH1VH4 C IgG

Heavy chain

kb. 85 gene kb. 27 gene kb. 6 gene

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The structure of antibody heavy chain

VL1 VL2 VLnVL4VL3 JL1 CJLnJL3JL2

JL3VL2 C kappa light chain

approx. 35 kappa gene approx. 5 kappa gene

approx. 30 lambda gene approx. 4 lambda gene

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The structure of antibody light chain

B cell

Antibody

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Production of antibodies in B cells

Recombination Junctional diversity

Somatic hipermutation

B cell

Antigene/epitope

Plazma cell Specific antibody

BCR

YY

YY

B cell

Clonal selection

Clonal expansion

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Clonal selection and clonal expansion

Y

YY

YB cell

B cell

B cell

antibody antigene

epitope

antibody

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Polyclonal antibodies

B cell

antigeneepitope

antibody

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Monoclonal antibodies

Spleen cell isolationHGPRT

antibody production

Myeloma cellsHGPRT

antibody production

Mouse immunization

Antigene

Fusion of spleen and myeloma cells, generation of hibridoma cells

Culturing of the hibridoma cells

antibody isolationY

YYY

Y Y

Y

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Production of antiodies in hybridoma cells

Mouse antibody Human antibody

Humanized antibody / chimera antibody

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Humanized antibodies

Mouse immunoglobulin gene

Human immunoglobulin gene

Human or humanized antibody production

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Production of human antibodies in genetically modified mice

5 db p9

5 db p75 db p3

5 db p6

2700 db p8

DNS - 6.4 kb

M13 bacteriophage

E. coli

900 nm

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The structure of M13 phage

F-pilus

Immobilized protein / affinity matrix

Specific elution

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Specific elution of immobilized phage particles

matrix

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Enzyme phage display

matrix

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Substrate phage display I.

matrixmatrix

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Substrate phage display II.

matrix

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Enzyme-substrate phage display I.

matrixmatrix

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Enzyme-substrate phage display II.

Phagemid

Recombinant phagemid

Various sequences

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Generation of phage libraries

hGH gene Protease substrate M13 gIII gene

phagemid vector

hGH gene Protease substrate M13 gIII gene

phagemid vector

Generation of various sequences

Phage library

Protease substrate sequence

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Generation of protease substrate phage library

hGH receptor

matrix

Protease

low pH

Protease resistent sequences

Protease sensitive sequences

Sequencing Protease

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Substrate phage display –engineering of protease substrate sequences

Intravenous injection of phage library

Phage particles bind to the vascular endothelial cell surface proteins

Biopsy

Removal of bound phages

Propagation of bound phages

Identification of phage- bound proteins/peptides

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In vivo phage display – mapping vascular endothelial cells

Limfocytes

mRNA

cDNA

antibody specific primer

Whole blood(immunized donor)

antibody genes

phagemid

E. coli cells

contain 108 differnt antibody genes

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Generation of antibody libraries from whole blood

Y Y YY

YY

Y

Tumor cellKiller cell (NK

cell or monocyte)

Antibody against tumor cells

Fc receptor

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The mechanism of antibody dependent cell mediated cytotoxicity (ADCC)

TNFalpha

IL2 receptoralpha chain

Inhibition of organ rejection after transplantation, especially in case of kidney transplantations.

• Psoriasis• Rheumatoid arthritis• Crohn disease• Spondilitis

Human-mouse chimera antibody

Monoclonal antibody • Adalimumab• Infliximab • Golimumab• Cetrolizumab pegol

Basiliximab

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Administration of therapeutic antibodies with immunosupressant activity

Bispecific antibody(approx. 300 kDa)

IgG - scFv

(Fab – scFv)2

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Forms of therapeutic antibodies

Fv

Tandem scFv Diabody Triabody Bispecific antibody

F(ab’)2Fab

scFv

S-S

scFv2dsFvS-S

Nanobody

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Forms of small-sized therapeutic antibodies